Not applicable
In many types of cancer cells, differentiation antigens are expressed. These antigens have been used as targets in cancer therapy. For example, CD19, CD20, CD22 and CD25 have successfully been used as targets in hematopoietic malignancies (Press, et al. New Eng. J. Med. 329:1219-1224 (1993); and Osterborg, et al., J. Clin. Oncol. 15:1567-1574 (1997)). However, this targeted cancer therapy has not been successful with solid tumors, in large part because the targeted antigens are also expressed in tissues from which the tumors arose. Thus, such targeted therapies kill healthy cells as well as the malignant cells.
In the United States, despite therapy, an estimated 15,000 women die of ovarian cancer each year. Although less common than ovarian cancer, mesotheliomas are known to be resistant to all chemotherapeutic agents and therefore have a high mortality rate. Because of the morbidity of these cancers, new therapeutic approaches to these malignancies are needed.
Common to ovarian, squamous cell and some stomach cancers as well as mesotheliomas is the expression of mesothelin on the cell surface (Chang, et al., Cancer Res. 52:181-186 (1992); Chang, et al., J. Surgical Pathology 16:259-268 (1992); and Chang, et al., Nat""l Acad. Sci. USA 93:136-140 (1996)). Mesothelin is a 40 kD GPI-linked glycoprotein antigen present on the surface of mesothelial cells. It is synthesized as a 69 kD precursor which is then proteolytically processed. The 30 kD amino terminus is secreted and has been termed megakaryocyte potentiating factor (Yamaguchi, et al. J. Biol. Chem. 269:805-808 (1994)). The 40 kD carboxyl terminus remains bound to the membrane as mature mesothelin (Chang, et al, Nat""l Acad. Sci. USA 93:136-140 (1996)). Unlike many cell surface antigens present on cancer cells, the membrane-bound form of mesothelin cannot be detected in the blood of cancer patients and is not shed by cultured cells into medium (Chang, et al., Cancer Res. 52:181-186 (1992)). In addition to malignant cells, mesothelin is also found on the cell surface of cells of mesothelial origin, including ovarian cancers. Because damage to cells in these tissues would not lead to life-threatening consequences, the presence of mesothelin on the surface of cancer cells makes it a promising candidate for targeted therapies.
Immunotoxins are antibodies directed against cell surface antigens joined to a toxic moiety. In the treatment of cancer, the antibody preferably is directed against a cell surface antigen expressed only on cancer cells. However, if the death of normal cells which also express the surface antigen is not more life-threatening than the existence of the malignancy, antibodies directed against cell surface antigens expressed on non-malignant cells can be used in cancer therapy. The toxic moiety of the immunotoxin can be any toxin that is not harmful to non-targeted cells at low concentrations after systemic administration. Such a toxin is the Pseudomonas aeruginosa exotoxin (PE). Previous studies with PE have demonstrated that the active portion of the protein is composed of domain II and III, both of which are located at the carboxyl end of the toxin.
The antibodies that target the immunotoxin can be polyclonal, monoclonal, or recombinant antibodies, such as chimeras or variable region fragments. If the antibody is non-recombinant, the immunotoxin must be formed by chemical conjugation of the antibody to the toxic moiety. If the antibody is produced recombinantly, the antibody can be joined to the toxin through chemical bonding or through recombinant fusion. In recombinant fusion, cDNA encoding the antibody is inserted, in frame, into a plasmid that already contains cDNA which encodes the toxin. Of course, the reverse could be done as well; the toxin cDNA can be inserted into a plasmid carrying cDNA which encodes the antibody.
Because of the potential large size of the immunotoxin, it is sometimes desired to join only a fragment of an antibody to the toxic moiety. Fab, FAbxe2x80x2 and F(ab)2 fragments can be made from polyclonal, monoclonal and chimeric antibodies and then joined to the toxin through chemical bonding.
Alternatively, a cDNA can be produced in which the variable regions of an antibody are connected to essential framework regions. These smaller antibodies are then secreted as double chain Fv antibodies or, if the heavy and light chain regions are joined either directly or through a peptide linker, as single chain Fv antibodies (scFv).
One method of creating a scFv is through phage display libraries made from splenic mRNA of mice immunized with an immunogen (Chowdhury, et al., Mol. Immunol. 34:9-20 (1997)). However, if a protein immunogen is naturally found in mammals but is recombinantly expressed in prokaryotes, the protein will not have the correct glycosylation pattern and may not have the correct conformation. Antibodies developed by the mouse in response to this immunogen may not recognize the protein in its native state. One solution to this problem is to immunize animals with the native protein made in mammalian cells, but purification from mammalian cells of sufficient amounts of some proteins, in particular cell surface proteins, may not be possible. Another solution, although not as common, is to immunize animals with cDNA which encodes the immunogen. The cDNA, under the control of an appropriate promoter, is introduced into the animal. After boosting injections and when the antibody titer reaches a maximum, the animals are sacrificed and the spleens removed to create the phage display library.
There is a need for better chemotherapeutic agents to control cancers such as ovarian cancer and mesotheliomas, both of which are rarely cured by currently available chemotherapies. The following disclosure describes such a chemotherapeutic agent.
In one aspect, the present invention relates to an anti-mesothelin antibody with a dissociation constant of less than 3xc3x9710xe2x88x928 M and which specifically binds to mesothelin on the surface of cells. In one embodiment, the anti-mesothelin antibodies are a single chain antibody comprising a variable heavy chain region and a variable light chain region. In yet other embodiments, the CDRs of the antibody are as indicated in FIG. 1. In other embodiments, the antibody is linked to an effector molecule, for example, a detectable label or a therapeutic agent. In one embodiment, the therapeutic agent is a toxin, preferably Pseudomonas exotoxin A or cytotoxic fragments thereof.
In another embodiment of the invention, the antibody is produced by immunizing an animal with cDNA which encodes mesothelin, preparing a phage display library from the mRNA isolated from the spleen of the immunized animal, selecting for phage that specifically bind mesothelin with a dissociation constant of less than 3xc3x9710xe2x88x928 M and which binds to mesothelin expressed on the surface of cells, isolating the nucleic acid from the bound phage, introducing the nucleic acid into a cell which expresses the phage-derived-mesothelin antibody and isolating the antibody from the cell. It further is contemplated that the nucleic acid sequences which encode the anti-mesothelin antibody are fused in frame to nucleic acid sequences which encode for the toxic moiety.
In another aspect, the present invention relates to an immunoconjugate of an anti-mesothelin antibody with a dissociation constant of less than 3xc3x9710xe2x88x928 M and which specifically binds to mesothelin on the surface of cells and a therapeutic agent or a detectable label. In one embodiment, the anti-mesothelin antibody is a single chain antibody comprising a variable heavy chain region and a variable light chain region. In other embodiments, the CDRs of the antibody are as indicated in FIG. 1. In yet another embodiment, the variable heavy chain region and the variable light chain region are bonded through a linker peptide. In other embodiments, the therapeutic agent is a toxin, preferably Pseudomonas exotoxin A or cytotoxic fragments thereof. Particularly preferred is PE38. In yet other embodiments, the variable heavy chain region of the antibody is peptide bonded to the carboxyl terminus of the therapeutic agent or detectable label.
In another aspect, the present invention relates to expression cassettes encoding either a recombinant anti-mesothelin immunoconjugate or a recombinant anti-mesothelin antibody. In some embodiments, the antibody is a single chain Fv antibody comprising a variable heavy chain region and a variable light chain region. In yet other embodiments, the CDRs of the antibody are as indicated in FIG. 1. In some embodiments, the immunoconjugate comprises a detectable label. In other embodiments, the anti-mesothelin antibody is bonded to a therapeutic agent, preferably a toxin and more preferably a Pseudomonas exotoxin A or cytotoxic fragments thereof, and most preferably PE38.
In yet another aspect, the present invention relates to host cells comprising expression cassettes which encode recombinant immunoconjugates or anti-mesothelin antibodies. In some embodiments, the host cells comprise an anti-mesothelin single chain Fv antibody comprising a variable heavy chain region and a variable light chain region. In yet other embodiments, the CDRs of the antibody are as indicated in FIG. 1. In further embodiments, the variable heavy chain region and the variable light chain region are linked through a peptide linker. The immunoconjugate comprises either a detectable label or a therapeutic agent bonded to an anti-mesothelin scfv fragment. In preferred embodiments, the therapeutic agent is a toxin, more preferably Pseudomonas exotoxin A or cytotoxic fragments thereof, and most preferably PE38.
In yet another aspect, the present invention relates to a method for inhibiting the growth of a malignant cell which expresses mesothelin on its cell surface. The method comprises the steps of contacting the malignant cell with an effective amount of a recombinant immunoconjugate comprising a toxic peptide bonded to an anti-mesothelin antibody which has a dissociation constant of less than 3xc3x9710xe2x88x928 M and binds to mesothelin expressed on cell surfaces. In one embodiment, the anti-mesothelin antibody is a scFv antibody with a variable heavy chain region and a variable light chain region. In another embodiment, the CDRs of the antibody are as indicated in FIG. 1. In yet other embodiments, the variable heavy chain region and the variable light chain region are linked by a peptide linker. In some embodiments the toxic peptide is Pseudomonas exotoxin (PE) or a cytotoxic fragment thereof, preferably PE38. In one embodiment, the variable heavy chain region is peptide bonded at the carboxyl terminus of the toxin. In some embodiments, the malignant cell is contacted with the immunoconjugate in vivo. The malignant cell, for example, can be an ovarian, squamous, gastric cell or a mesothelioma.
In a further aspect, the present invention is directed to a method for detecting the presence of mesothelin in a biological sample. The method comprises the steps of contacting the biological sample with an anti-mesothelin antibody which has a dissociation constant of less than 3xc3x9710xe2x88x928 M and binds to mesothelin expressed on cell surfaces, and allowing the antibody to bind to mesothelin under immunologically reactive conditions, wherein detection of the bound antibody indicates the presence of the mesothelin. In one embodiment, the antibody is a scFv fragment comprising a variable heavy (VH) region and a variable light (VL) region. In yet other embodiments, the CDRs of the antibody are as indicated in FIG. 1. In another embodiment, the VH region and the VL region are linked through a peptide linker. In one embodiment, the antibody employed in the method is detectably labeled. In vet other embodiments, the antibody is conjugated to a toxic peptide and the presence of the immunoconjugate is detected by antibodies to the toxic peptide. In some embodiments, the method is personnel in vivo in a mammal.
In yet a further aspect, the present invention is directed to pharmaceutical compositions comprising the immunoconjugates of this invention. In another aspect, the present invention is directed to kits which can be used to detect mesothelin on cell surfaces.